title: Orai3 TM3 point mutation G158C alters kinetics of 2-APB-induced gating by disulfide bridge formation with TM2 C101.
abstract: After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. However, in the absence of Ca(2+) store depletion and STIM interaction, the mammalian homologue Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. The "slow" phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by variable reversal potentials. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS), but in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that is sensitive to the actions of 2-APB.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697.
authors.forename: Anna
authors.initials: A
authors.lastname: Amcheslavsky
authors.forename: Olga
authors.initials: O
authors.lastname: Safrina
authors.forename: Michael D
authors.initials: MD
authors.lastname: Cahalan
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D005998:Glycine / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary
chemical_list: D001896:Boron Compounds; D015220:Calcium Channels; D004220:Disulfides; C522867:Orai3 protein, human; C109986:2-aminoethoxydiphenyl borate; D003545:Cysteine; D000409:Alanine; D005998:Glycine